The present invention relates to novel combinations of non-competitive folic acid analogues with protecting agents, and to methods of treatment using these combinations.
European Patent Application 0505640 provides a method for improving the therapeutic utility of GAR-transformylase inhibitors and other antifolates by co-administering a folate binding protein binding agent to the host undergoing treatment.
Thymidylate synthase is an enzyme catalysing the terminal step in the de novo synthesis of thymidylate required for DNA synthesis. It has been postulated that inhibitors of this enzyme may be expected to have anti-tumour activity and it has been reported (Jones et al, J. Med. Chem. 1986, 29, 468) that the in-vivo antitumour activity of N10-propargyl-5,8-dideazafolic acid arises solely from its inhibitory effect on this enzyme.
PCT publication WO/91/19700 discloses the compound of the formula (I) 
or a salt thereof, wherein the dotted line represents a single or double bond.
R1 is C1-4 alkyl or amino optionally substituted by a C1-4 alkyl C1-5 alkanoyl or benzyl group;
R2, R3, R4 and R5 are the same or different and each is selected from hydrogen, phenyl, halo, nitro.
a group S(O)n R8 wherein n is the integer 0, 1 or 2 and R8 is halo or is C1-4 alkyl or a group NR9R10 wherein R9 and R10 are both hydrogen,
a group NR11R12 wherein R11 and R12 are the same or different and each is hydrogen or C1-4 alkyl.
a group OR13 wherein R13 is hydrogen or C1-4 alkyl optionally substituted by halo;
a C1-4 aliphatic group optionally substituted by a group OR14 or NR14R15 wherein R14 and R15 are the same or different and each is hydrogen or C1-4 alkyl:
or two of R2 to R5 are linked together to form a benzo group.
or one of R2 to R5 is a group xe2x80x94Xxe2x80x94Yxe2x80x94R16 wherein X is CH2, NR17, CO or S(O)m and m is 0, 1 or 2 and R17 is hydrogen or a C1-4 aliphatic group and Y is CH2, O, NR17xe2x80x2 or S(O)mxe2x80x2 wherein mxe2x80x2 is 0, 1 or 2 and R17 is hydrogen or a C1-4 aliphatic group provided that X and Y are only the same when each is CH2, or xe2x80x94Xxe2x80x94Yxe2x80x94 is a group xe2x80x94Oxe2x80x94. xe2x80x94NR17xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94 or xe2x80x94Nxe2x95x90Nxe2x80x94 wherein R17 is as hereinbefore defined, R16 is a C1-4 aliphatic group or a 5- or 6-membered aromatic ring optionally substituted by a group R18 at a position at least one carbon atom removed from that linked to Y, the 5- or 6-membered ring being optionally further substituted by a halo atom; and R18 is halo, C1-4 alkoxy, nitro, nitrile, C1-4 alkyl optionally substituted by halo, halo or a group COR19 wherein R19 is hydroxy, C1-4 alkoxy or C1-6 alkyl optionally substituted by one or two carboxyl groups or C1-12 esters thereof or R19 is a group NR20R21 wherein R20 and R21 are the same or different and each is hydrogen or C1-4 alkyl optionally substituted by hydroxy or R19 is an amino acid group or an ester thereof in which the first nitrogen atom of the amino acid group may be linked to the 5- or 6-membered aromatic ring to form a further 5- or 6-membered heterocyclic ring or R19 is an C2-3 alkylene croup linked to the 5- or 6-membered aromatic ring to form a further 5- or 6-membered ring;
R6 and R7 are the same or different and each is hydrogen, C1-4 alkyl optionally substituted by hydroxy or C1-4 alkoxy or together form a benzo group;
provided that at least one of R2 to R7 is other than hydrogen and that R4 is not methoxy when R1 is hydroxy or methyl is an inhibitor of the enzyme thymidylate synthase and has anti-tumour activity.
By the term halo is meant fluoro, bromo, chloro and iodo.
By the term C1-4 aliphatic group is meant a C1-4 alkyl, alkenyl, or alkynyl group.
By the term amino acid group is meant naturally occurring amino acids.
Preferred amino acid groups include glycine, glutamic acid and polyglutamic acid groups.
When the amino acid group is linked to the 5- or 6-membered aromatic ring, this is via a carbon atom of the aromatic ring adjacent to carbon to which COR19 is attached.
Preferably the dotted line is a double bond.
Suitable substituents for the aromatic ring R16 include halo, C1-4 alkyl and C1-4 alkoxy each optionally substituted by one to five halo atoms. Most suitably there are one or two substituents selected from fluoro, chloro, methyl, trifluoromethyl and methoxy, and preferably fluoro, or no substituents on the aromatic ring. In one preferred embodiment, xe2x80x94Xxe2x80x94Yxe2x80x94R16 is a group 
wherein R18 is as hereinbefore defined and preferably a group COR19 as hereinbefore defined and R22 is hydrogen or fluoro.
In a further preferred embodiment Xxe2x80x94Yxe2x80x94R16 is a group 
wherein H2NR19a is a glutamic or polyglutamic acid group and Z is CH2, S or O.
Suitably, R1 is an amino group optionally substituted by one or two methyl or ethyl groups or R1 is a methyl or ethyl group. Preferably R1 is an amino or methyl group.
Suitably, at most only three, and preferably at most only two, of R2 to R5 are other than hydrogen and each is independently selected from hydrogen, halo, hydroxy, nitro, C1-3 alkyl optionally substituted by hydroxy or C1-2 alkoxy, C1-3 alkoxy, amino optionally substituted by one or two methyl or ethyl groups, or a group S(O)n R23 wherein n is 0, 1 or 2 and R23 is a C1-4 alkyl group or an amino group optionally substituted by one or two methyl or ethyl groups, or one of R2 to R5 is a group xe2x80x94Xxe2x80x94Yxe2x80x94R24 where R24 is a group 
wherein R18, R19a, R22 and Z are as hereinbefore defined. In one preferred embodiment R18 is nitro or a group 
wherein R25, R26 and R27 are the same or different and each is hydrogen or a C1-4 alkyl group and t is an integer from 0 to 6. Preferably R25, R26 and R27 are hydrogen and t is 0. Preferably Z is CH2 or S.
Preferably one of R2 to R5 is a group xe2x80x94Xxe2x80x94Yxe2x80x94R24 as hereinbefore defined. Preferably R3 is a group xe2x80x94Xxe2x80x94Yxe2x80x94R24.
Suitably R6 and R7 are the same or different and each is hydrogen, methyl, ethyl or methyl substituted by bromo, hydroxy or methoxy. Preferably R7 is hydrogen and R6 is methyl.
Preferably xe2x80x94Xxe2x80x94Yxe2x80x94 is a group xe2x80x94SO2NR17xe2x80x94 or CH2NR17 wherein R17 is as hereinbefore defined.
Suitably R17 is hydrogen or a C1-4 alkyl or alkenyl group and preferably R17 is hydrogen or methyl.
A further group of compounds of the formula I is that of the formula (II) 
or a salt thereof, wherein R1, R6, R7 and the dotted line are as hereinbefore defined and R28 to R31 are the same or different and each is selected from hydrogen, halo, nitro, a group S(O)nR8, a group NR11R12, a group OR13, or a C1-4 aliphatic group optionally substituted by a group OR14 or NR14R15 wherein R8, R11, R12, R13, R14 and R15 hereinbefore defined, provided that R28 to R31 are not all hydrogen and that R30 is not methoxy wherein R1 is hydroxy or methyl.
A preferred group of compounds of the formula (I) is that of the formula (III): 
or a salt thereof, wherein R1, R6 and R7 are as hereinbefore defined and R32 to R35 are the same or different and one is a group Xxe2x80x94Yxe2x80x94R16 and the others are the same or different and each is selected from hydrogen, halo, nitro, a group S(O)nR8, a group NR11R12 a group OR13 or a C1-4 aliphatic group optionally substituted by a group OR14 or NR14R15, wherein X, Y, R8, R11, R12, R13, R14, R15 and R16 are as hereinbefore defined.
A further preferred group of compounds of the formula (I) is that of the formula (IV): 
wherein R1, R6, R7 and R32 to R32 are as hereinbefore defined.
Preferably R33 is a group Xxe2x80x94Yxe2x80x94R16 as hereinbefore defined.
Preferred compounds of the formula (I) include:
3-Amino-9-bromobenzo[f]quinazolin-1(2H)-one
3-Amino-9-ethynylbenzo[f]quinazolin-1(2H)-one
N-(4-((3-Amino-1,2,5,6-tetrahydro-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamic acid
N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamic acid
N-(4-((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamic acid
N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamic acid
N-(4(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glumatic acid
(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino-1-oxo-2-isoindolinyl)glutaric acid
9-((4-Acetylanilino)methyl)-3-methylbenzo[f]quinazolin-1(2H)-one
3-Methyl-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one
N-(4-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glutamic acid
3-Amino-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one
9-((4-Acetylanilino)methyl)-3-aminobenzo[f]quinazolin-1(2H)-one
(RS)-2-(2-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-2-oxoethyl)glutaric acid
Ethyl4-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyrate
4-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyric acid
N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycine
Ethyl N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycinate
Certain compounds of the formula (I) contain asymmetric carbon atoms and are, therefore, capable of existing as optical isomers. The individual isomers and mixtures of these are included within the scope of the present invention.
It has now been found that the compounds of formula (I) can be used in combination with a protecting agent described hereinafter to prevent or decrease unwanted side effects of these compounds.
The present invention relates to the administration of the compounds of formula (I) in combination with a protecting agent which blocks the intestinal toxicity of the compounds of formula (I) without blocking their activity. Suitable protecting agents are folates, for example, folic acid, tetrahydrofolate, 5-formyltetrahydrofolate (leucovorin) or 5-methyltetrahydrofolate. Other protecting agents include thymine or thymidine. The protecting agent may be administered as an oral formulation. It is within the scope of the invention to administer the protecting agent before, during or after the administration of a compound of formula (I). Preferably, the protecting agent will be given slightly before administration of a compound of formula (I), i.e. between 15 minutes and one hour before the compound of formula (I).
The present invention also relates to combination therapy comprising the administration of non-competitive folic acid analogues in combination with protecting agents as defined above. This allows the use of the non-competitive folic acid analogues at higher doeses than could be used in the absence of the protecting agent as defined above, and therefore higher, more potent doses can be achieved while minimizing host toxicity. The presence of the protecting agent reduces the toxicity of the non-competitive folic acid analogues at the doses utilized in the absence of the protecting agent (i.e. less toxicity at the same dose in the presence of a protecting agent).
Non-competitive folic acid analogues are defined as structural analogues of folic acid (including but not limited to the benzoquinazoline compounds of formula I, e.g., compounds of example 1 or 2) expressing classical non-competitive inhibition kinetics (vs. the folate substrate) on the target enzyme (e.g., thymidylate synthase, glycineamide ribonucleotide transformylase or dihydrofolate reductase). Alternatively, the non-competitive inhibitors of the present invention are defined as structural analogues of folic acid (as defined above) which retain their tumour cell kill in the presence of the folate protecting agents defined above, on the target enzyme. The non-competitive inhibitor may been given as an intravenous or intraperitoneal bolus or infusion and the protecting agent may be given as an intravenous or intraperitoneal bolus infusion or as an oral formulation. For a definition of classical non-competitive inhibition see I. H. Segal (1975) Enzyme kinetics, John Wiley and Sons, which is incorporated herein by reference.
More specifically, protecting agents selected from folic acid, tetrahydrofolate. 5-formyltetrahydrofolate (leucovorin) or 5-methyltetrahydrofolate can block the intestinal toxicity of compounds of formula (I) without blocking their anti-tumor activity.
Preferably the non-competitive, folic acid analogue (i.e., non-competive inhibitor) is a compound of formula (I) and the protecting agent is folic acid or 5-formyltetrahydrofolate. More preferably, the non-competitive, folic acid analogue (non-competitive inhibitor) is a compounds selected from (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid. N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamic acid, or N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamic acid and the protecting agent which is used in combination is folic acid or 5-formyltetrahydrofolate. Most preferably, the compound (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxoxbenxo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid is utilized in combination with folic acid or 5-formyltetrahydrofolate.
The salts of the compounds of the present invention can also be used in combination with the protecting agent or salts thereof.
Suitable salts of the compounds of the present invention or of the protecting agents may comprise acid addition salts derived from an amino group or anionic species derived from a compound of formula (I) or protecting agent, for example when this is substituted by a carboxy group, and a cation. In both types of salts, the therapeutic activity resides in the moiety derived from the compounds defined herein and the identity of the other component is of less importance although for therapeutic and prophylactic purposes it is, preferably, pharmaceutically acceptable to the patient. Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic and methanesulphonic and arylsulphonic, for example p-toluenesulphonic, acids. Examples of salts comprising an anionic species derived from a compound of formula (I) and a cation include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth salts, such as magnesium and calcium salts and salts formed with organic bases, for example, amino salts derived from mono-, di- or tri-(lower alkyl) or (lower alkanol)amines, such as triethanolamine and diethylamino-ethylamine, and salts with heterocyclic amines such as piperidine, pyridine, piperazine and morpholine. The pharmaceutically acceptable salts together with the salts which are not thus acceptable have utility in the isolation and/or the purification of the compounds of the invention, and the pharmaceutically unacceptable salts are also useful in being convertible to the pharmaceutically acceptable salts by techniques well known in the art.
The route by which the compound or salt of the present invention is administered to the animal may be oral, topical, parenteral (including subcutaneous, intradermal, intramuscular, intravenous or rectal). If the compound or salt is presented in the form of a pharmaceutical formulation, which is preferred, then the actual formulation employed will of course depend on the route of administration elected by the physician or veterinarian. For example, if oral administration is preferred, then the pharmaceutical formulation employed is, preferably, one which is suitable for such a route.
A therapeutically effective amount of a compound or salt of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of a non-competitive folic acid analogue of the present invention (as described hereinbefore) to be used in combination with a protecting agent (as described hereinbefore) for the treatment of neoplastic growth, for example colon or breast carcinoma will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day would usually be from 70 to 3500 mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se.
An effective amount of a compound of the present invention for the treatment of disorders of the immune system (e.g. thermatoid arthritis and tissue rejection) and related diseases such as psoriasis, will generally be in the range of 0.5-10 mg/kg body weight of recipient (mammal) per day. Thus for a 70 kg adult human, the actual amount per day would usually be from about 5-700 mg per day and this amount may be given in a single dose per day or more usually dosing would be intermittent e.g. 12 hourly intervals or weekly. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se.
An effective amount of a compound of the present invention for the treatment of bacterial and fungal infections is in the range of 0.1-100 mg/kg bodyweight of recipient (mammal) per day and preferably in the range of 1-50 m/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day is from 70-3500 mg and this amount may be given in a single dose per day or more preferably in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se.
An effective amount of a protecting agent (e.g., folic acid) of the present invention for use in combination with a non-competitive, folic acid analogue (including but not limited to the compounds of the present invention as described herein) is in the range of 1-300 mg/kg bodyweight of recipient (mammal) per day and preferably in the range of 5-50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, the actual amount per day is from 350-3500 mg and this amount may be given in a single dose per day or more preferably in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same.
The treatment of neoplastic growth with a compound of the present invention may at times require the administration to the animal of an antidote or rescue agent, e.g. thymidine.
The compounds, per se, of the present invention which can be used in combination with a protecting agent as described herein include, but are not limited to, those disclosed in PCT publication WO/91/19700. The protecting agents of the present invention are readily available and well known to those skilled in the art.
Whilst it is possible for the compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical formulation.
Accordingly, the present invention further provides a pharmaceutical formulation, for medicinal application, which comprises a compound of the present invention and a protecting agent or pharmaceutically acceptable salts thereof, as hereinbefore defined, in combination with one or more pharmaceutically acceptable carriers therefor, and optionally one or other therapeutic agredients.
The pharmaceutical formulation may optionally contain other therapeutic agents that may usefully be employed in conjunction with the compound or salt of the present invention, for example a pyrimidine nucleoside transport inhibitor that is capable of enhancing the antineoplastic activity of the compounds and salts of the presents invention. The expression xe2x80x9cpharmaceutically acceptablexe2x80x9d as used herein in relation to the carrier is used in the sense of being compatible with the compound or salt of the invention employed in the formulation and with any other therapeutic agent that may be present, and not being detrimental to the recipient thereof. The carrier itself may constitute one or more excipients conventionally used in the art of pharmacy that enable the compound or salt of the present invention and any other therapeutic agent that may be present, to be formulated as a pharmaceutical formulation.
The pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration although the most suitable route will probably depend upon, for example, the condition and identity of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods will known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e., the compound or salt and protecting agent of the present invention, with the carrier.
In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation. The pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units, such as a capsule, cachet, tablet, or lozenge, each containing a predetermined amount of the active ingredient, as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid such as a syrup, elixir or a draught, or as an oil-in water liquid emulsion or a water-in-oil liquid emulsion. The formulation may also be a bolus, electuary or paste.
Generally, a tablet is the most convenient pharmaceutical formulation suitable for oral administration. A tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating agent and/or a surface active agent. Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient.
The pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqeuous sterile injection solutions which may contain, for example, an anti-oxidant, a buffer, a bacteriostat and a solution which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
The pharmaceutical formulations of the present invention suitable for rectal administration may be presented as a suppository containing, for example, cocoa butter and polyethylene glycol.
Compounds and salts of formula (I) have anti-neoplastic activity in the human colon SW480 adenocarcinoma cell culture cytotoxicity tests in which representative compounds of the present invention have been shown to be active, and in human breast MCF7 adenocarcinoma cell culture. It has thus been established that compounds of the present invention are able to inhibit neoplastic growth. Therefore, compounds and salts of the present invention are of use in medicine and in particular in the treatment of neoplastic growth, including solid tumours such as melanoma breast and colon tumours in mammals. Accordingly, the present invention yet further provides a method for the treatment of susceptible malignant tumours and leukemia in a animal. e.g., a mammal, which comprises administering to the animal a therapeutically effective amount of a compound or salt of the present invention in combination with a protecting agent. In the alternative, there is also provided a compound or salt of the present invention in combination with protecting agent. In the alternative, there is also provided a compound or salt of the present invention in combination with a protecting agent for use in medicine and in particular for use in the treatment of a neoplastic growth, e.g., malignant tumours.
The present invention also provides the use of a compound of formula (I) or a salt therof in combination with a protecting agent for the manufacture of a medicament for the treatment of neoplastic growth.
The animal requiring treatment with a compound or salt of the present invention in combination with a protecting agent is usually a mammal, such as a human being.
The following examples illustrate the preparation and pharmacological properties of representative compounds which are useful in the present invention and which demonstrate the invention. These examples are illustrations only and should not be construed as limiting or narrowing the scope of the invention.